Arabian Journal of Chemistry (2020) 13, 7851–7859

King Saud University

Arabian Journal of Chemistry

www.ksu.edu.sawww.sciencedirect.com

ORIGINAL ARTICLE

Cross-mixing study of a poisonous Cestrum species,

Cestrum diurnum in herbal raw material by

chemical fingerprinting using LC-ESI-QTOF-MS/

MS

* Corresponding authors at: H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, Unive

Karachi, Karachi 75270, Pakistan (S.G. Musharraf). Pharmacognosy Group, Department of Medicinal Chemistry, Biomedical Centre, U

University, Box 574, 75 123 Uppsala, Sweden (H.R. El-Seedi).

E-mail addresses: hesham.el-seedi@farmbio.uu.se (H.R. El-Seedi), musharraf1977@yahoo.com (S.G. Musharraf).

Peer review under responsibility of King Saud University.

Production and hosting by Elsevier

https://doi.org/10.1016/j.arabjc.2020.09.0161878-5352 � 2020 The Authors. Published by Elsevier B.V. on behalf of King Saud University.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Hamna Shadab a, Muhammad Noman Khan a, Faraz Ul Haq a, Hamad Ali b,

Hesham R. El-Seedic,d,*, Syed Ghulam Musharraf

a,b,*

aH.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi,Karachi 75270, PakistanbDr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological

Sciences, University of Karachi, Karachi 75270, PakistancPharmacognosy Group, Department of Pharmaceutical Biosciences, BMC, Uppsala University, SE-751 23 Uppsala, Sweden.d International Research Center for Food Nutrition and Safety, Jiangsu University, Zhenjiang, 212013, China

Received 28 July 2020; accepted 9 September 2020

Available online 23 September 2020

KEYWORDS

LC-ESI-MS/MS;

Chemical fingerprinting;

Quantification;

Adulteration;

Cytotoxicity

Abstract Poisonous plants are widely distributed and may have risk of phytotoxicity upon mixing

with medicinal plants. Several species of Cestrum genus are poisonous and linked with many serious

health issues. In the present study, cross-mixing of a toxic plant, Cestrum diurnum with morpholog-

ically resembling medicinal plant, Adhatoda vasica was studied using chemical fingerprinting

approach. LC-ESI-MS/MS tool was used to develop the chemical fingerprints of three toxic species

of Cestrum, including, C. diurnum, C. nocturnum and C. parqui. Total forty-three compounds were

identified using high-resolution LC-ESI-MS/MS data comparison. Chemometric analyses were

done to compare the distribution of identified compounds present in these Cestrum species. One

of the identified compounds, nornicotine (a toxic compound) was also quantified using LC-IT-

MS/MS. Adulteration study was conducted by mixing toxic C. diurnum in A. vasica with various

ratios (w/w) and five differentiable compounds were identified to detect the adulteration. The

method was able to detect up to the limit of 5% mixing of toxic C. diurnum. Moreover, cytotoxicity

rsity of

ppsala

7852 H. Shadab et al.

of the methanolic extracts of these three species were also studied on normal human PBMC (periph-

eral blood mononuclear cells) and all found to be toxic, while the C. nocturnum showed the highest

level of toxicity with the IC50 12.5 lg/mL.

� 2020 The Authors. Published by Elsevier B.V. on behalf of King Saud University. This is an open access

article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Many plant species contain toxic components and thus can beeither fatal or can cause serious harm when consumed. Thetoxic constituents of these plants, have been reported in severalstudies using isolation of toxic compounds, chromatographic

fingerprinting and mass spectrometric screening (Carlieret al., 2015). Cestrum species are cultivated and widely usedas ornamental plants in South East Asian countries. Several

species of Cestrum are also known for their severe toxicityand ingestion of these plants can cause illnesses such as vom-iting or diarrhea (Filmer and Dodge, 2012). When consumed,

C. diurnum triggers dystrophic calcinosis of cardiac tissues,arteries, and tendons. Different toxic alkaloids have beenreported in these species (Halim et al., 1971). It also containsa steroidal glycoside which is hydrolyzed to vitamin D3 and

causes increased intestinal absorption of calcium beyond thelevels that can physiologically be accommodated (Mair andLove, 2012). C. parqui is also reported to be toxic showing hep-

atotoxic effects when consumed (Cullen and Stalker, 2016).The global trade of herbal medicines has been growing by

about 15% yearly and the Southeast and South Asian coun-

tries are the largest source of raw material for these products(Ming and Sulaiman, 2016). The fast-growing global trade ofherbal products also increased the concerns for their safety

and quality. The raw herbal material which is used in the pro-duction of herbal products is quite hard to identify physicallyin its dried state. So there is possibility of adulteration of anytoxic or non-toxic plants with medicinal plants used in herbal

products, as there is lack of proper quality control and stan-dardization protocols in the production of these products(Mbendana et al., 2019; Shaheen et al., 2019). Adulteration

in raw plant material can cause serious consequences on healthand safety of the consumers which can be countered by devel-oping sensitive and accurate quality control protocols (Srirama

et al., 2017). In literatures, many analytical tools are men-tioned to detect mixing/adulteration in plant material, includ-ing electrophoric techniques, DNA-based tools (genomics),

differential scanning calorimetry (DSC), chromatographictools, fluorescence and vibrational spectroscopy, isotopicdetection, elemental tools, NMR spectroscopy, mass spec-trometry, liquid chromatography mass spectrometry (LC–

MS and LC–MS-MS) (Witjaksono and Alva, 2019). Amongstall, LC–MS and LC–MS-MS are preferred choice for complexsamples due to high-throughput and sensitive analysis.

In previously reported studies, identification, and quantifi-cation of some metabolites in extracts of C. diurnum, C. parquiand C. nocturnum were done through mass spectrometric tech-

niques (Black et al., 2017; Chaskar et al., 2017; Hughes et al.,1977; Doshi, 2016; Hanhineva et al., 2011). This study aims todevelop a method that can help to prevent the adulteration oftoxic Cestrum species. For this purpose, two-step strategy was

conducted, firstly, comprehensive chemical profiles of three

species of Cestrum genus were developed. Secondly, an adul-teration study of A. vasica was performed with physically

resembling toxic species C. diurnum. A. vasica is a well-known medicinal plant used in many herbal products for thetreatment of fever, cough and asthma (Rahman et al., 2019).

Due to their similar appearance, there is a strong chance ofA. vasica being adulterated with toxic C. diurnum. The aerialparts of A. vasica and C. diurnum are shown in Fig. 1. Further-

more, the cytotoxic effects of all three Cestrum species wereevaluated in human peripheral blood mononuclear cells(PBMCs). These profiles would be useful in the preventionof their adulteration and the quality control of herbal

formulations.

2. Experimental

2.1. Chemicals and reagents

For the preparation of sample solutions and mobile phase,HPLC-grade methanol was acquired from Merck (Darmstadt,Germany) while Ultrapure Type-I water was obtained from

the Ultra-pure Water Purifier (GenPure, Waltham, MA.,USA). Formic acid (>98% pure) was obtained by DaeJungChemical and Metals (South Korea).

2.2. Sample collection

C. diurnum, C. parqui and C. nocturnum samples were gatheredfrom different regions of Pakistan including Islamabad, Swat,

Lahore, Bajour and Karachi (Supplementary Table 1). Theidentification of plant samples was carried out by plant tax-onomist, Mr. Shabbir Ijaz, Herbarium, University of Karachi.

2.3. Sample preparation

Plant samples were ground, and 1 g of each sample was trans-

ferred to a conical centrifuge tube (15 mL) containing 10 mL ofmethanol. Extraction was done through sonication for 30 minat ambient temperature which was followed by centrifugation

at 6000 RPM for 30 min. 1 mL supernatant from each cen-trifuge tube was filtered through a syringe-driven PTFE filter(0.22 lm) into another centrifuge tube (1.5 mL). For analysis,all the samples were transferred into HPLC vials and twenty

times diluted with methanol.

2.4. UHPLC-ESI-QTOF-MS/MS analysis

Chemical fingerprinting of three plant species was performedusing high-resolution Bruker maXis-II QTOF Mass Spectrom-eter (Bremen, Germany) coupled to Thermofisher Ultimate

3000 series Ultra Performance Liquid Chromatography.Macherey-Nagel C-18 column (3.0 � 50 mm, 1.8 mm particle

Fig. 1 Aerial parts of Adhatoda vasica (left) and Cestrum diurnum (right).

Cross-mixing study of a poisonous Cestrum species, Cestrum diurnum in herbal raw material 7853

size) was selected for chromatographic separation. Linearmobile phase gradient system was applied, consisting of

type-I water as eluent (A) and methanol as eluent (B), with0.1% formic acid as additive in both mobile phases. Solventgradient was run as 40% B in 0.0 to 1.0 min, 50% B in 1.0

to 2.0 min, 60% B in 2.0 to 7.0 min, 80% B in 7.0 to7.5 min, 90% B in 7.5 to 9.0 min and then again 40% B in9.0 to 9.5 min. The overall run-time was 10 min including

0.5 min of column equilibration at the end. The constant sol-vent flowrate was set at 0.7 mL/min and 2 lL of each samplewas injected through autosampler. The column was main-tained at the temperature of 40 �C. Each experiment was

accompanied with calibration using sodium formate solution(10 mM). Mass detection range was set between 50 and1500 m/z. For positive ionization mode, 4500 V of capillary

voltage was provided while drying gas (nitrogen) was flownat the rate of 10 mL/min with a temperature of 300 �C. Fornegative ionization mode, all the parameters were same as with

positive ionization mode except for the capillary voltage whichwas set at 3500 V.

A smart strategy was designed for targeted and untargetedidentification of metabolites. The targeted identification was

done by generating a custom-made library of compoundsreported from these plants. Bruker Daltonics Target Analysis1.3 (Bremen, Germany) was used to screen the high-

resolution mass spectra for these reported compounds bycomparing accurate masses and isotopic patterns. The untar-geted identification was performed by utilizing different ESI

MS/MS libraries such as Mass Bank of North America, NISTMS/MS libraries, and Mass Bank of Europe. All these librariesare easily accessible, and these libraries were incorporated in

the NIST MS search system to make searching simple. Theparameters like exact masses, isotopic patterns and MS/MS

fragmentations were used for identification. The thresholdvalue for high-resolution m/z matching was set under 5 ppmerror and for isotopic matching, it was set under 50 mSigma

value. DataAnalysis (version 4.4) was utilized to generateExtracted Ion Chromatograms (EIC) of each identifiedcompound.

2.5. Quantitation using HPLC-ESI-IT-MS/MS

Quantitation experiments were performed on Bruker ama-Zon speed Ion trap mass spectrometer (Bremen, Germany)

coupled to Thermofisher Scientific Ultimate 3000 High-Performance Liquid Chromatography system (USA). HPLCsystem was accompanied with a quaternary pump, a column

thermostat, and an auto sampler. Macherey-Nagel C-18 col-umn (3.0 � 50.0 mm, 1.8 mm particle size) was selected forchromatographic separation. Mobile phase contained water

(solvent A) and methanol (solvent B), both having 0.1% for-mic acid as additive. The linear gradient system used wasjust same as for the profiling experiment. A narrow spectral

scan range 50–500 m/z was set to achieve good sensitivity.All the voltages were optimized for maximum resolutionand were then set as; endplate offset at 500 V and nozzlevoltage at 4500 V. Ion Charge Control (ICC) was set as

20,0000. Drying gas pressure was flown with a rate of4.0 L/min, 49.9 �C temperature and 10.0 psi pressure. Theaccumulation time was set at 200 ms while an average of

5 spectra was fixed. Six calibration solutions were preparedranging 1–2000 ng/mL concentration.

7854 H. Shadab et al.

For method validation, three QC samples were preparedwith concentrations 25, 175 and 375 ng/mL. QC samples wereanalysed with six replicates each for intra-day and inter-day

analysis. The percent relative standard deviation (%RSD) val-ues were used to determine precision while percent accuracywas used to determine accuracy.

2.6. Preparation of adulterated samples

Shade-dried samples of A. vasica (medicinal plant) and toxic

plant C. diurnum were ground to a fine powder and mixed atdifferent ratios. Total, nine powdered plant samples were pre-pared, 10 g each, with 1%, 2%, 5%, 10%, 20%, 30%, 40%,

50% and 60% mixing of toxic C. diurnum. The remaining sam-ple preparation strategy was same as for the chemicalfingerprinting.

2.7. Cytotoxicity assay of Cestrum species

Human peripheral blood mononuclear cells (PBMCs) wereextracted from the whole blood using a gradient density cen-

trifugation technique utilizing Ficoll-hypaque (Sigma, USA).10 mL of the venous whole blood sample was collected froma healthy volunteer in a heparinised tube. The blood was

gently layered on 5 mL Ficoll medium in 15 mL tube and cen-trifuged for 20 min at 400g with breaks off. The cells in theinterphase (buffy coat) representing PBMCs were softly aspi-rated out and transferred aseptically into sterile centrifuge

tubes. The isolated cells were cleaned with 10 mL sterile 1xPBS (phosphate buffer saline) solution at 200g for 10 min toremove the platelets. Trypan Blue dye exclusion method was

used to count the PBMCs.Isolated PBMCs were cultured in RPM1 (Roswell Park

Memorial Institute)-1640 medium (Gibco, UK) supplemented

with 10% FBS (Fetal bovine serum) (Gibco, USA) 1% Pen/Strep (Gibco, Germany) at 37 �C and 5% CO2 in humidifiedconditions. PBMCs were seeded in 96 well plates at density

4 � 104 cells per well having 100 lL complete culture media.The cells were dosed with methanolic extracts of CN, CPand CD at 200, 100, 50, 25, 12.5, 6.25 and 3.12 lg/mL, andDMSO (Sigma, USA) as vehicle control. The treated cultures

were incubated for 48 h at 37 �C and 5% CO2 in moistenedconditions.

The effects of C. nocturnum, C. parqui and C. diurnum

extracts on cell viability were determined using Alamar Blue(Resazurin). Briefly, 10 lL of 0.02% Alamar blue (Thermo sci-entific, USA) solution was added to post-treatment PBMCs

cultured in 96 well plates and incubated for 8 h in a CO2 incu-bator at 37 �C. The fluorescence readings were measured at560 nm excitation and 590 nm emission wavelengths, using

Thermofisher Scientific Varioskan LUX multimode microplatereader and SkanIt tool version 4.1. The half-maximal inhibi-tory concentration (IC50) values were calculated, and the per-cent cell viability was used to determine the cytotoxic effects of

tested extracts. The data was analysed through GraphPadPrism v6.01 through two-way ANOVA and Bonferroni P-value test for multiple comparisons and represented as

mean ± SD, n = 3. <0.05P-value was believed to be statisti-cally substantial. Independent Ethics Committee, InternationalCenter for Chemical and Biological Sciences has approved the

study protocols under protocol number ICCBS/IEC-022-HB-2019/Protocol/1.0.

2.8. Compliance with ethics requirements

Independent Ethics Committee working under InternationalCenter for Chemical and Biological Sciences has approved

all the study protocols under protocol number ICCBS/IEC-022-HB-2019/Protocol/1.0. All procedures followed were inaccording to Helsinki Declaration of 1975 (revised 2008).

Informed permission was taken from volunteer before involv-ing in the research.

3. Results and discussion

3.1. Chemical fingerprinting and identification of compounds

Chromatographic conditions were optimized for good shapeand well separated peak. The overall runtime along with equi-libration was kept within 10 min while the solvent flowrate was

fixed at 0.7 mL/min. The LC-MS chromatograms of each Ces-trum species collected from different regions of Pakistan wereobserved quite similar except for some variations in peak

intensities (Supplementary Fig. 1). A total of forty-three plantmetabolite were identified by comparing accurate masses, frag-mentation data and isotopic pattern (Table 1). The MS/MS

spectra of these compounds are given in SupplementaryTable 2. Most of the identified compounds belong to car-boxylic acids, esters, and flavonoids classes of compounds.

3.2. Chemometric analysis

Heatmap cluster analysis of identified compounds was per-formed using software Perseus (1.6.2.1) to study the distribu-

tion of these compounds in all the samples. For heat maps,log2(X) transformed peak areas were used. Identified com-pounds were screened in all the twenty plant samples using

Target Analysis and Data Analysis acquisition software. Heat-map clusters showed good similarities in the spread of com-pounds in plant samples growing at different regions.

Heat-map cluster analysis demonstrates a clear spread ofcompounds in selected Cestrum species (Fig. 2). Heatmapshowed nornicotine (compound 09) present in all samples of

all three species with no significant difference in intensities.The identified compounds were mostly found in C. diurnumsamples including compounds 12, 13, 15, 18, 27, 32 and 39,contrarily compound 20, 34 and 42 are specific in Cestrum noc-

turnum while no compound was found specific to C. parquisamples only. These compounds are important in chemicalprofiles as they are differentiative. The clustering has shown

clear grouping of all the samples related to each species exceptsample CP2. This split in clustering of C. parqui samples couldbe attributed to variation in metabolites due to growing condi-

tions, genotype and age of plant (Verma and Shukla, 2015).SIMCA version 14.1 (Umetrics, Sweden) was used to pro-

duce principal component analysis (PCA plot) according topeak areas of found compounds. The discrimination between

the three species was determined. All the C. diurnum sampleswere clearly discriminated against with other samples of C.nocturnum and C. parqui lied quite separated in the plot with

Fig. 2 Heatmap cluster of identified compounds in all samples.

Cross-mixing study of a poisonous Cestrum species, Cestrum diurnum in herbal raw material 7855

clear discrimination between them. The PCA plots of positive

and negative ionization modes data are given SupplementaryFig. 2, respectively. Venn diagrams were also created employ-ing Umetrics SIMCA 14.1 (Sweden), to show the possible rela-

tionships between these three different groups of Cestrumspecies based on mutually identified compounds. The Venndiagrams are given in Supplementary Fig. 3.

3.3. Quantification of nornicotine

From the identified compounds, nornicotine was selected forquantitation in C. diurnum, C. parqui and C. nocturnum sam-

ples as it is one of the mutually identified toxic compoundsof these plants (Nishtha and Rao, 2017) There are manyreported cases of neurotoxicity and cardiotoxicity caused by

nornicotine (Holtman et al., 2010; Kovacic and Thurn, 2005;Stairs et al., 2007; Sundaragiri and Tandur, 2016). Nornicotinewas quite stable and showed good mass spectrometric features.

Chemical structure of nornicotine, MS/MS fragments and cal-ibration curve are given in Supplementary Fig. 4. A good chro-matographic separation is necessary for LC-MS analysis,

which was optimized using a reverse phase (C-18) column.The overall runtime was limited to 8 mins with equilibration.Positive ionization mode mass spectrometry was found morecompatible with nornicotine as it showed better sensitivity

than negative ionization mode. A good shaped and intenseparent ion peak was observed at 0.45 min and selected forMS/MS transitions. 149.0 m/z and 132.0 m/z were selected as

quantifier ions and to get good intensity peaks, fragmentoramplitude was optimized and set at 90 V. Nornicotine wasfound abundant in all the samples and its concentration (mg/

Kg) is given in Supplementary Table 3. The concentration of

nornicotine is in the range of 10.04 to 11.27 mg/Kg in collectedsamples of Cestrum species. The developed quantitationmethod has shown good accuracy (98.7 to 103.0%) and repro-

ducibility (under 4.8 %RSD). Supplementary Table 4 showsthe results of the method validation experiment.

3.4. Adulteration studies using LC-ESI–QTOF-MS/MS

Chemical profiles of C. diurnum and A. vasica were generatedusing LC-ESI-QTOF analysis with the same conditions of pos-itive ionization mode. Detailed chemical profiles of A. vasica

along with quantitation of bioactive metabolites have alreadybeen published (Rahman et al., 2019). The chemical profilesof both plants were compared and the region of 3 min to

6.5 min was aimed due to presence of C. diurnum related peaks.Five marker compounds were selected based on (i) visible peakchromatogram in C. diurnum, (ii) absent in A. vasica and (iii)

identified compounds. These compounds are 5-hydroxy-2-[2-hydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]-7,8-dimethoxychromen-4-one (A) at

2.8 min., ladroside (B) 3.4 min., plumbagine at 3.9 min. (C), 7-hydroxy-4-(methoxymethyl)coumarin (D) at 3.9 min. and 1-(b-D-Glucopyranosyloxy)-1,4a,5,6,7,7a-hexahydro-5-hydroxy-7-methyl-6-[[(2E)-1-oxo-3-phenyl-2-propen-1-yl]oxy]-, methyl

ester, (1S,4aS,5S,6R,7R,7aR)-cyclopenta[C]pyran-4-carboxylic acid (E) at 5.1 min. (Table 1). Their extracted ionschromatograms and fragment ions are shown in Supplemen-

tary Fig. 5. Profiles of adulterated samples with different per-cent mixing of C. diurnum were also produced and their peakareas were plotted to check the lowest detectable percentage

Table 1 List of identified compounds in positive and negative modes of ionization.

Codes Compound names Formula R.

T

Adduct m/z

Observed

m/z

calculated

Error

(ppm)

MS/MS

fragments

1 3a-Galactobiose C12H22O11 0.5 [M + H]+ 365.1059 365.1054 1.3 347.0963,

203.9517,

185.0409

2 Glycan 3a-Galactobiose C12H22O11 0.5 [M + H]+ 365.1067 365.1054 3.5 347.0963,

203.9517,

185.0409

3 Methyl (1S,4aS,5R,7S,7aS)-1-(b-D-

glucopyranosyloxy)-5,7-dihydroxy-7-

methyl-1,4a,5,6,7,7a -hexahydrocyclopenta

[C]pyran-4-carboxylate

C17H26O11 0.6 [M + H]+ 429.1372 429.1376 2.1 267.0839,

235.0568

4 Lamiide C17H26O12 0.6 [M + Na]+ 445.1322 445.1322 0.0 445.1523,

233.0420,

265.0691 ,

283.0802

5 Sinapinic acid C11H12O5 0.7 [M + H]+ 225.0763 225.0757 2.6 147.0422,

174.0534,

192.0647

6 Genipin C11H14O5 0.7 [M + H]+ 227.0919 227.0912 3.0 165.0542,

175.0386

7 6,8-Dihydroxy-7-methoxy-3-methyl-3,4-

dihydroisochromen-1-one

C11H12O5 0.8 [M + H]+ 225.0756 225.0754 3.9 179.0687,

147.0422

8 Acteoside C29H36O15 1.4 [M + H]+ 647.1951 647.1946 0.7 163.0398,

467.1526

9 Nornicotine C10H12N2 0.4 [M + H]+ 148.2184 148.2181 0.6 132.0

10 2-Hydroxy-2-(4-methoxyphenyl)-1-

methylethyl hexopyranoside

C16H24O8 1.7 [M + Na]+ 367.1369 367.1360 2.4 185.0410,

203.0670

11 1-(b-D-Glucopyranosyloxy)-1,4a,5,6,7,7a-

hexahydro-6-[[(2E)-3-(4-hydroxy-3-

methoxyphenyl)-1-oxo-2-propen-1-yl]oxy]-

7-methyl-, (1S,4aS,6S,7R,7aS)- cyclopenta

[C]pyran-4-carboxylic acid

C26H34O13 2.7 [M + Na]+ 553.1921 553.1916 0.9 337.1311,

195.0641,

177.0529

12 5-Hydroxy-3-[4-hydroxy-2-

[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-

(hydroxymethyl)oxan-2-yl]oxyphenyl]-7-

methoxychromen-4-one

C22H22O11 2.7 [M + Na]+ 485.1063 485.1067 0.8 323.0500

13 Phenylalanine C9H11NO2 0.6 [M + H]+ 166.0820 166.0859 2.3 152.1058

14 Scoparone C11H10O4 2.8 [M + H]+ 207.0657 207.0652 2.4 179.0700

15 5-Hydroxy-2-[2-hydroxy-3-

[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-

(hydroxymethyl)oxan-2-yl]oxyphenyl]-7,8-

dimethoxychromen-4-one (A)a

C23H24O12 2.8 [M + Na]+ 515.1165 515.1160 0.9 353.0626,

185.0421

16 Oenin C23H25O12 2.8 [M]+ 493.1341 493.1336 1.0 331.0808,

316.0568

17 Petunidin-3-O- b-glucoside C22H23O12 3.3 [M]+ 479.1189 479.1189 0.0 317.0651

18 Ladroside (B)a C26H32O13 3.4 [M + H]+ 575.1740 575.1724 2.7 373.1321,

177.0529,

195.0641

19 2-Methoxy-1,4-naphthoquinone (C)a C11H8O3 3.9 [M + H]+ 189.0551 189.0488 3.3 187.0910,

161.0571

20 7-Hydroxy-4-(methoxymethyl)coumarin

(D)aC11H10O4 2.8 [M + H]+ 207.0657 207.0652 2.4 147.0436,

161.0583,

157.0388,

192.0415

21 Coumarin-6-carboxaldehyde C10H6O3 4.0 [M + H]+ 175.0399 175.0395 2.2 147.0436

22 4-Hydroxynaphthalene-1,2-dione C10H6O3 4.0 [M + H]+ 175.0399 175.0387 4.8 147.0439

23 1-(b-D-Glucopyranosyloxy)-1,4a,5,6,7,7a-

hexahydro-5-hydroxy-7-methyl-6-[[(2E)-1-

oxo-3-phenyl-2-propen-1-yl]oxy]-, methyl

ester, (1S,4aS,5S,6R,7R,7aR)- Cyclopenta

[C]pyran-4-carboxylic acid (E)a

C26H32O12 5.1 [M + H]+ 559.1799 559.1795 0.8 357.1346,

225.1083,

277.0912,

195.0642,

177.0530,

149.0602

24 Protopine C20H19NO5 6.0 [M + H]+ 354.1333 354.1336 0.8 336.1234,

7856 H. Shadab et al.

Table 1 (continued)

Codes Compound names Formula R.

T

Adduct m/z

Observed

m/z

calculated

Error

(ppm)

MS/MS

fragments

265.0380,

223.0837,

206.0810

25 Sphinganine C20H30O 8.5 [M + H]+ 287.2688 287.2697 1.7 270.2474,

226.2160

26 Benzyl dodecyl-dimethyl ammoniumb C21H38N 8.4 [M + H]+ 304.2991 304.2998 0.2 212.2368

27 Malvidin 3-O-b-D-glucoside C12H25O12 2.8 [M + H]+ 493.1341 493.1341 1.0 331.0808,

315.0368

28 3-(2,4-Dihydroxypentyl)-8-hydroxy-6-

methoxyisochromen-1-one

C22H23O12 0.6 [M + H]+ 295.1131 295.1176 1.7 277.1020,

259.1020

29 4-Hydroxynapthalene-1,2-dione C10H6O3 4.0 [M + H]+ 175.0384 175.0387 1.7 147.0439

30 Indole-3-lactic acid C11H11NO3 0.8 [M + H-

H2O]+188.0707 188.0706 0.5 146.0606,

170.0602

31 Benzoylcholine C12H18NO2 0.6 [M]+ 208.1324 208.1327 0.8 149.0571

32 Temazepam C16H13ClN2O2 2.1 [M + H]+ 301.0820 301.0738 2.7 283.0715,

255.0763

33 S-Hexylglutathione C16H29N3O6S 4.5 [M + Na]+ 414.1655 414.1669 3.3 396.1532

34 Shanzhiside methyl ester C17H26O11 0.7 [M + H]+ 406.1553 406.1548 1.2 195.0639,

177.0530,

167.0682,

149.0570

35 5-Hydroxy-2-methyl-7-[(2S,3R,4S,5S,6R)-

3,4,5-trihydroxy-6-(hydroxymethyl)oxan-

2-yl]oxychromen-4-one

C16H18O9 0.6 [M + H]+ 355.1014 355.1024 2.8 194.0483,

193.0457

36 Methyl (1S,4aS,6R,7R,7aR)-1-(b-D-

glucopyranosyloxy)-5,6-dihydroxy-7-

methyl-1,4a,5,6,7,7a-hexahydrocyclopenta

[C]pyran-4-carboxylate

C17H26O11 0.8 [M + FA-

H]�451.1459 451.1479 4.4 405.1396,

243.0875

37 Acetocide C29H36O15 1.4 [M�H]� 623.1977 623.1981 0.6 461.1659,

179.0351,

161.0243

38 Methyl (1S,4aR,6S,7R,7aS)-1-(b-D-

glucopyranosyloxy)-4a,6,7-trihydroxy-7-

methyl-1,4a,5,6,7,7a-hexahydrocyclopenta

[C]pyran-4-carboxylate

C17H26O12 0.8 [M + FA-

H]�467.1409 467.1406 0.6 403.1248,

331.1045,

259.0825,

241.0717,

179.0563

39 Methyl 1-(hexopyranosyloxy)-5,6-

dihydroxy-7-({[(2E)-3-(4-hydroxyphenyl)-

2-propenoyl]oxy}methyl)-1,4a,5,6,7,7a-

hexahydrocyclopenta[C]pyran-4-

carboxylate

C26H32O14 2.8 [M�H]� 567.1714 567.1720 1.0 387.1077,

303.0866,

179.0346,

163.0400

40 2-(3,5-Dihydroxy-4-methoxyphenyl)-5,7-

dihydroxy-3-[(2S,3R,4R,5R,6S)-3,4,5-

trihydroxy-6-methyloxan-2-yl]

oxychromen-4-one

C22H22O12 3.1 [M�H]� 477.1035 477.1038 0.6 315.0510

41 Petunidin-3-O- b-glucoside C22H22O12 3.1 [M�H]� 477.1035 477.1047 2.5 315.0510

42 9-Hydroxy-10E,12Z-octadecadienoic acid C18H32O3 9.6 [M�H]� 295.2274 295.2284 3.3 277.2165,

171.1026

43 12(13)-Epoxy-9Z-octadecenoic acid C18H32O3 9.6 [M�H]� 295.2274 295.2278 1.3 277.2165,

195.1387,

171.1026

44 Lactitol C12H24O11 1.8 [M�H]� 343.1289 343.1247 1.2 181.0815

a = Marker compounds selected for adulteration studies.b = Contaminant plasticizer.

Cross-mixing study of a poisonous Cestrum species, Cestrum diurnum in herbal raw material 7857

of C. diurnum in the adulterated sample (Fig. 3). The compar-ative profiles showed that the developed method was able to

detect as low as 5% mixing of C. diurnum in medicinal plantA. vasica.

3.5. Comparative cytotoxic effect of Cestrum species on PBMCs

The cytotoxic effects of crude methanolic extracts of three Ces-trum species were determined against PBMCs isolated from a

Fig. 4 (A-C) Cytotoxic effects of different concentrations of CN (C. nocturnum), CP (C. parqui) and CD (C. diurnum) methanol extract

on human PBMCs at 48 h. (D) Comparison between the cytotoxic effects of CN, CD and CP indicate that 12.5 to 50 lg/mL

concentrations of CN significantly decrease the PBMCs viability as compared to CD and CP. The data represented as mean ± SD, n = 3.

*P value < 0.05 **P value < 0.01, ***P < 0.001 vs control. ###P value < 0.001 CN vs CP. +++P value < 0.001 CN vs CD.

Fig. 3 Positive ionization base peak chromatogram (BPC) of Cestrum diurnum and Adhatoda vasica mixed at different ratios.

7858 H. Shadab et al.

healthy donor, at concentrations range of 3.12–200 lg/mL. A

noteworthy decrease in cell viability was observed in treatedPBMCs in dose-dependent way which indicates that extractsfrom all three species of Cestrum (C. diurnum, C. parqui and

C. nocturnum) were highly cytotoxic. The extract of C. noctur-

num showed a high level of cytotoxicity and significantly

induced cell death at a concentration � 6.25 lg/mL withIC50 12.5 ± 1.4 lg/mL (Fig. 4A). Extract of C. diurnum signif-icantly inhibited cells viability at � 12.5 lg/mL with IC50

28 ± 3.3 lg/mL (Fig. 4B), while C. parqui extract displayed

Cross-mixing study of a poisonous Cestrum species, Cestrum diurnum in herbal raw material 7859

cytotoxic effects toward PBMCs at � 25 lg/mL with IC50 29.3 ± 3.5 lg/mL (Fig. 4C). Moreover, the C. nocturnum wasfound significantly more toxic at 12.5, 25 and 50 lg/mL as

compared to C. diurnum and C. parqui. However, no statisti-cally substantial variation was found among cytotoxic effectsof C. diurnum and C. parqui extracts at any concentration as

shown in Fig. 4D.

4. Conclusion

A rapid and sensitive method was developed using high-resolution LC-QTOF-MS/MS analysis for chemical profilingand identification of secondary metabolites in three toxic spe-

cies of Cestrum and to study the adulteration of a toxic plantwith the herbal plant material. Phytotoxicity of methanolicextracts of three Cestrum species were observed PBMCs iso-

lated from a healthy donor. Different chemometric means wereutilized to study the spread of forty-three identified com-pounds in multiple samples of all three species for the develop-ment of characteristic fingerprint. The phytochemical profiles

developed would also be helpful in taxonomical identification,toxicological studies and to study the bioactive components ofthese plants. Similarly, adulteration protocol will be very help-

ful to prevent accidental consumption of this toxic species andhelp to develop quality control methods.

Acknowledgments

The authors acknowledge the support of Organization for theProhibition of Chemical Weapons (OPCW) for research fund-

ing under project number S/1258/2015. The authors are alsograteful to Mr. Saeedur Rahman and Mr. Shabbir Ijaz forsample collection and Mr. Junaid Ul Haq and Mr. Arsalan

Tahir for technical help.

Declaration of Competing Interest

The authors declare no conflict of interest

Appendix A. Supplementary material

Supplementary data to this article can be found online athttps://doi.org/10.1016/j.arabjc.2020.09.016.

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